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Sedat, J.

Publications and source records attributed to Sedat, J..

5 recordsLinked to original sources

A Proposed Unified Mitotic Chromosome Architecture

A molecular architecture is proposed for an example mitotic chromosome, human Chromosome 10. This architecture is built on a previously described interphase chromosome structure based on Cryo-EM cellular tomography (1), thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle, for mitotic chromosomes, is specific coiling of the 11-nm nucleosome fiber into large scale approximately 200 nm structures (a Slinky (2, motif cited in 3) in interphase, and then further modification and subsequent additional coiling for the final structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well known cytological configurations. In addition, proof is experimentally provided, by digital PCR technology, that G1 T-cell nuclei are diploid, thus one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils. Significance StatementThe significance of this proposed mitotic chromosome architecture is that a specific, sequenced chromosome, human Chromosome 10, can be built into a specific architecture that accounts for the dimensional values and cytological descriptions, a first time result. Since this molecular architecture is an extension of the interphase chromosome structure, a coiling of the 11-nm nucleosome fiber with further coiling, a unifying molecular structure motif is present throughout the entire mitotic cycle, interphase through mitosis.

cell biology↗

A Proposed Unified Interphase Nucleus Chromosome Structure: Preliminary Preponderance of Evidence

Cellular cryo-electron tomography (CET) of the cell nucleus using Scanning Transmission Electron Microscopy (STEM) and the use of deconvolution (DC) processing technology has highlighted a large-scale, 100-300 nm interphase chromosome structure (LSS), that is present throughout the nucleus. This chromosome structure appears to coil the nucleosome 11-nm fiber into a defined hollow structure, analogous to a Slinky (S) (1, motif used in 2) helical spring. This S architecture can be used to build chromosome territories, extended to polytene chromosome structure, as well as to the structure of Lampbrush chromosomes. Significance StatementCryo-preservation of the nuclear interior allows a large scale interphase chromosome structure--present throughout the nucleus--to be seen for the first time. This structure can be proposed to be a defined coiled entity, a Slinky. This structure can be further used to explain polytene chromosome structure, an unknown chromosome architecture as well as for lampbrush chromosomes. In addition, this new structure can be further organized as chromosome territories, using all 46 human interphase chromosomes as an example, easily into a 10 micron diameter nucleus. Thus, interphase chromosomes can be unified into a flexible defined structure.

cell biology↗

Entropy Regularized Deconvolution of Cellular Cryo-Transmission Electron Tomograms

Cryo-electron tomography (cryo-ET) allows for the high resolution visualization of biological macromolecules. However, the technique is limited by a low signal-to-noise ratio (SNR) and variance in contrast at different frequencies, as well as reduced Z resolution. Here, we applied entropy regularized deconvolution (ER DC) to cryo-electron tomography data generated from transmission electron microscopy (TEM) and reconstructed using weighted back projection (WBP). We applied DC to several in situ cryo-ET data sets, and assess the results by Fourier analysis and subtomogram analysis (STA).

biophysics↗

3D Deconvolution Processing for STEM Cryo-Tomography

The complex environment of biological cells and tissues has motivated development of three dimensional imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryo-scanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles, and then in representative cryo-tomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin. Significance statementElectron tomography is used to reveal the structure of cells in three dimensions. The combination with cryogenic fixation provides a snapshot in time of the living state. However, cryo-tomography normally requires very thin specimens due to image formation by conventional phase contrast transmission electron microscopy (TEM). The thickness constraint can be relaxed considerably by scanning TEM (STEM), yet three-dimensional (3D) reconstruction is still subject to artifacts inherent in the collection of data by tilted projections. We show here that deconvolution algorithms developed for fluorescence microscopy can suppress these artifacts, resulting in significant contrast enhancement. The method is demonstrated by cellular tomography of complex membrane structures, and by segmentation of chromatin into distinct, contiguous domains of heterochromatin and euchromatin at high and low density, respectively.

biophysics↗

CryoSIM: super resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultra-structural imaging

Rapid cryo-preservation of biological specimens is the gold standard for visualising cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo electron microscopy or cryo soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly open source Python control software. Therefore, cryoSIM democratises the ability to detect molecules using super-resolution fluorescence imaging of cryo-preserved specimens for correlation with their cellular ultrastructure.

cell biology↗